AquaFlux™ Model AF200

(a) What is TEWL ?

(b) How do you measure TEWL ?

You cannot measure TEWL directly because that would imply measuring water flux inside the SC. Instead, TEWL is measured indirectly, by measuring the evaporation flux in the adjacent air. Of course, this only works as long as TEWL is the ONLY source of evaporation flux, ie skin surface must be dry and there must be no sweating.

Illustrated on the left are three instruments (evaporimeters) that are commonly used for measuring TEWL. They use three different measuring methods as described below.

(i) Open-chamber Method

(ii) Condenser-chamber Method

(iii) Unventilated-chamber Method

References

The AquaFlux™ out-performs all its competitors in terms of accuracy, sensitivity, repeatability, reproducibility and versatility. Below, we present data on three aspects, namely (a) how do they correlate, (b) how do the readings compare and (c) where’s the difference.

(a) How do they correlate ?

(b) How do the readings compare ?

Although the measurements correlate well, the readings generally differ. This is due to calibration differences, with no agreement among manufacturers about best practice. The calibration disagreement in the above study was found to be quite small, ~14%, but disagreements by a factor 2 or more are not uncommon. Such disagreements can be resolved by the traceable droplet calibration method developed in collaboration with the UK National Physical Laboratory.

(c) Where’s the difference ?

Signal fluctuations cause scatter in TEWL measurements. One comparative test, for example, found AquaFlux™ TEWL repeatability to be more than ten times better (CV=1.4%) than comparable Evaporimeter repeatability (CV=16%) [2].

How this translates into practice is illustrated in the figure below, where two skin sites of the same volunteer were repeatedly tested in alternation.

References

The AquaFlux™ and the VapoMeter have one thing in common: they both use closed-chamber measurement methods. In every other respect, they are very different, right down to the measurement method itself. If it’s shirt pocket convenience you want, then forget about the AquaFlux™. If it’s performance you need, then forget about the VapoMeter.

A comparison between an AquaFlux™ Model AF102 and a Delfin VapoMeter was published as a poster at the ISBS Meeting in Philadelphia in 2005 [1]. Here we show a similar comparison with updates to illustrate the performance of our latest  AquaFlux™ Model AF200.

An in-vitro test can give you an important insight into how well an instrument is performing. Are the readings constant when the flux is constant ?

Below are the results of an experiment consisting of 200 repeat measurements using an upside-down wet-cup source with each instrument. More…

These data are characterised by a coefficient of variation of CV=0.93% for the AquaFlux™ and CV=10.3% for the VapoMeter. This gives a measure of what each instrument contributes to the scatter in any experiment, in-vivo or in-vitro.The bigger the instrumental scatter, the less reliance you can place on a single reading. According to Gaussian statistics, you would need to average ~100 VapoMeter readings to match the precision of a single AquaFlux™ reading.

Below is a table summarising the scatter of readings observed in an experiment consisting of 12 repeat measurements in rapid succession on 7 untreated sites of the volar forearm of an elderly volunteer. More…

The AquaFlux™ scatter shows a clear pattern across the 7 sites: lowest in the middle part of the forearm and rising towards either end. There is no such pattern in the VapoMeter data. There is also a significant difference in the magnitude of the observed scatter. The mean over all 84 measurements works out to CV=3.8% for the AquaFlux™ and CV=10.2% for the VapoMeter.From the above it is clear that the much lower instrumental scatter of the AquaFlux™ enables it to resolve small differences in skin properties, heterogeneity in this case. With the Vapometer, the measurements are dominated by instrumental scatter.

The two instruments work very differently.

The AquaFlux™ records ~2 flux readings per second. TEWL measurement speed depends on skin condition and software settings. Flux readings settle quicker with dry, well acclimatised skin than with moist skin. Software settings determine how precisely the flux readings need to settle before a TEWL measurement terminates. The default criterion is a standard deviation of 0.075 g/(sq.m h) in a running average over the last 10 flux readings. You can adjust these values to trade off precision for speed. There is no waiting time between measurements – you can site-hop.

The VapoMeter measures vapour accumulation rate rather than flux. A typical contact time of ~10 seconds is required to produce a reading. This is followed by a recovery period of up to 90 seconds, where the measurement chamber needs to be voided of accumulated water vapour before the next measurement can begin.

AquaFlux™ and VapoMeter measurement times were compared in an experiment consisting of 12 repeat measurements in rapid succession on 7 untreated sites of the volar forearm of an elderly volunteer. Average repeat-times worked out to ~47 seconds for an AquaFlux™ Model AF200 and 38 seconds for a VapoMeter.

(d) Quality control

How do you know that you are measuring TEWL and not a momentary sweat gland emission or surface evaporation from a minute quantity of superficial moisture? The VapoMeter just gives you a number. The AquaFlux™ gives you detailed information in its recorded flux curves.

You’ve finished a study and are pondering the results. You spot something unexpected and are not sure how to interpret it. With the AquaFlux™ you can inspect the recorded flux curves and other supporting data. With the VapoMeter you’re stuck. Which one gives you more insight into what went on? Which one may save you having to do a repeat-study?

(e) Versatility

References

TEWL measurement speed depends on several factors, including instrumental characteristics, skin condition and the accuracy you want.

The AquaFlux™ determines TEWL from water vapour flux density measurements performed at a rate of ~2 per second. When you first place the measurement head onto the skin, the conditions in the measurement chamber are perturbed and it takes some time for the instrumental readings to settle. From then on, the software is in control, looking for the end-point that meets your accuracy requirements.

Normal TEWL Measurement

Normal TEWL measurements on acclimatised, healthy skin are quick, because there is no skin surface water (SSWL) to slow things down. According to the TEWL guidelines for open-chamber instruments [1, 2], you need to wait for the probe to recover after every TEWL measurement before starting the next measurement. This is not necessary with the AquaFlux™. You can go from site to site without pause and get the job done with maximum efficiency.This in-vivo site-hopping technique was first tested with a now obsolete Model AF102 AquaFlux™ in a series of measurements on 7 sites of a volar forearm [3]. The updated measurement times using a Model AF200 AquaFlux™ were found to be as follows:-

Accuracy & Measurement Time

As indicated above, measurement time can be traded for accuracy. If speed is important, then you can set less stringent TEWL endpoint settings in the software. The effect of this is illustrated in the three screenshots below.

The main endpoint criterion used in the software is a user-specified Standard Deviation (StDev) over a user-specified number of the most recent flux density readings. This is indicated in the screenshots above by the cyan-coloured average bars, where the vertical length is ±1 StDev and the horizontal length indicates the data included in the average. The default settings for the AquaFlux™ are:-

StDev = 0.075 g/(sq.m h) over the 10 most recent flux density readings.

How this looks in practice is indicated in the uppermost screenshot. These settings were used for determining the measurement times in the table above.

If you want quicker measurements, then you can use a larger StDev setting. This is illustrated in the two lower screenshots with StDev settings of 0.3 and 0.5 g/(sq.m h). Clearly, the flux readings have not settled, but if you use the same settings throughout a study, then the relative TEWL values will be meaningful. These StDev values are typical for open-chamber instruments. The higher sensitivity of the AquaFlux™ makes them look inappropriate.

References

(a) Probe angle

Angular dependence may be an issue with TEWL instruments, because natural convection air movements arising out of the temperature difference between the skin and the ambient air may disturb the measurement. However, different instruments using different measurement methods are affected differently, as follows:-

According to published TEWL Guidelines [1, 2] open-chamber instruments such as the C+K Tewameter can only be used reliably on horizontal surfaces.

Unventilated-chamber instruments such as the Delfin VapoMeter have also been found to be affected by probe angle [3], although the manufacturer claims orientation-independence.

For the condenser-chamber AquaFlux™, detailed measurements show that the probe has an orientation-dependence that is non-symmetrical, depending on whether the RH and T sensors in the wall of the measurement chamber are above or below the chamber axis [4]. For negative angles, where these sensors are below the chamber axis, probe sensitivity was found to decrease by as much as ~6% relative to the calibrated value. For positive angles, where these sensors are above the chamber axis, probe sensitivity was found to remain within about ±1% of the calibrated value. The probe can therefore be used with all surface orientations with little effect on sensitivity, as long as its inclination is confined to the positive semi-circle.

(b) Contact Pressure

Increases of contact pressure between the skin and a TEWL measurement head may produce changes to the skin and changes to the measurement geometry. Changes to the skin include compression of the underlying tissues and surface stretching, with the skin around the periphery of the measurement chamber depressed and the skin within the measurement orifice raised into a convex spherical shape. Changes to the measurement geometry include changes in the relative positions of the sensors and a decreased separation between the sensors and the skin surface. Decreases of contact pressure may compromise the seal between the skin and a measurement head and cause leakage and positional drift.

In practice, relatively large contact pressure effects have been reported for open-chamber instruments [6, 7]. Guidelines [1, 2] recommend that the contact pressure of the probe onto the skin should be kept low and constant, with measurements within a series preferably performed by the same operator.

For the unventilated-chamber VapoMeter, De Paepe et al [5] observed no statistically significant change of TEWL readings (from 7±2 to 8±3[g/(m²h)]) when the contact pressure was increased.

The effect of contact pressure on condenser-chamber AquaFlux™ TEWL measurements was assessed in an experiment using a spring balance to measure contact force. Measurements of mid-volar forearm TEWL were repeated 18 times on the same site with contact forces progressively increased then reduced in the range 0.1 to 2kg. The average TEWL over all 18 measurements worked out to 8.23[g/(m²h)] with a CV of 1.7% [4]. It appears, therefore, that the effect of contact pressure on TEWL measurement has more to do with measurement head design than skin barrier property change.

(c) Atmospheric Pressure

TEWL measurement methods all rely on evaporimetry, where TEWL is inferred from the water evaporation flux in the air immediately adjacent to the skin surface. All the commonly used methods involve the diffusion water vapour through air, from the skin surface to the sensor(s) and beyond. The associated mass diffusion coefficient, according to gas theory, changes with temperature and pressure and this affects the calibration of TEWL instruments.

The effect on open-chamber TEWL measurements was first discussed by Nilsson [6]. He concluded that, at a given location, weather-related changes of atmospheric pressure could affect TEWL readings by as much as ±6%. This was deemed to be too small for further consideration.

The effect on condenser-chamber AquaFlux™ and unventilated-chamber VapoMeter instruments was investigated by Kramer er al [8]. For the AquaFlux™, measurements showed a clear trend of increasing TEWL readings with increasing atmospheric pressure (gradient = [150±22]x10^-4 [g/(m²h)]/hPa). No trend with pressure was apparent in the VapoMeter measurements (gradient = [13±44]x10^-4 [g/(m²h)]/hPa), although a weak dependence cannot be ruled out, given the relatively large standard deviation of the gradient.

(e) Probe Temperature

Probe temperature may be affected by hand heat as well as by ambient temperature. The guidelines for open-chamber instruments [1, 2] recognise probe temperature as an essential variable, with instrumental readings increasing with temperature until the probe and skin temperatures are similar. Their recommendation is not to touch the probe before and during measurements. Instead, they recommend to handle the probe with a burette clamp, its cable, a coating or by wearing gloves.

Unventilated-chamber VapoMeter readings have also been found to be strongly temperature dependent. By holding the VapoMeter between both hands, De Paepe et al [5] caused its temperature to increase by ~6°C and observed an increase of volar forearm TEWL readings from a baseline of 7±2[g/(m²h)] to 15±6[g/(m²h)], which works out to a temperature coefficient of ~1.3±1.1[g/(m²h)] per °C temperature change.

By contrast, the condenser-chamber AquaFlux™ probe shows little effect upon heating. A typical figure, calculated as the root-mean-square (RMS) average over 10 instruments, is ~0.05±0.06[g/(m²h)] per °C temperature change [4].

(g) Skin cooling

The heat flux caused by the low condenser temperature (conduction through the air & radiation) is too small to cause the skin surface temperature to change significantly. We went to some lengths to try to measure an effect, using highly sensitive thermocouples and superglue (PhD thesis, Don O’Driscoll, London South Bank University, 2001). The main finding was that skin cooling was dominated by conduction between the skin and the measurement head and any effect from the condenser was masked by this. No effect on measured TEWL values was found. Don has made a full recovery from his superglue ordeal.

(f) Skin drying

References

This is an end-of-the-day job, except for the wettest or most demanding applications.

Captured water vapour forms a layer of ice on the condenser. The thickness of this layer grows at the rate of ~26microns for every milligram (mg) of captured water vapour. To put this into context, with a typical volar forearm TEWL of 10g/(sq.m h), it would take over 2.5 hours of continuous measurement to accumulate 1mg of ice.

The main effect of ice build-up is a gradual change of calibration, attributable mainly to the reduction of measurement chamber length. This is illustrated by the measurements below, where ice was accumulated by repeatedly calibrating an AF200 instrument by the droplet method

The blue correlation line indicates a 1% change of calibration for every ~2.5mg of accumulated ice. This translates into ~500 volar forearm TEWL measurements at 10g/(sq.m h).In practice, you need to keep an eye on the Ice Monitor of the AquaFlux™ software. You normally leave the instrument running all day and get rid of the ice when the job is done. Before going home, you switch the instrument to stand-by mode and allow the ice to melt and the water to evaporate

If you do a lot of high-flux measurements, then you may need to stop for ~10 minutes to get rid of the ice. Switch to stand-by mode for a few seconds while you dab off the melt-water on the condenser using one of the fibre-free cleanroom swabs supplied with the instrument. When done, switch back on, close the measurement orifice and wait for the baseline to settle.

Why ?

Calibration is crucial. Use multiple instruments, share data with collaborators, relate what you did yesterday with what you plan for tomorrow, defend your claims in court – the list is endless. The alternative is normalisation, but that wastes information.

How Often ?

You should check the flux density calibration at 3-6 month intervals, or before a major study. You can do this yourself in about half an hour using the calibration accessories supplied with the instrument.

By Whom ?

By you of course! You can also get it done as part of a Passport Service, but that involves cost, shipping and delay. Calibration accessories (calibrated micro-syringe & calibration caps) are supplied as standard with the instrument, so what’s to stop you? The software handles all the calculations and you’re done in less than half an hour. At the end you have the choice to use the new calibration or to discard it, so nothing is lost if you don’t quite get it right.

How ?

References

The quick answer is yes and no. Yes, much of what they recommend is valid for all measurement methods. No, they were written for open-chamber instruments and the AquaFlux™ is different. If you want the long answer, read on.

There are two published guidelines for TEWL measurement in general [1, 2], and one for TEWL measurement in non-clinical settings [3]. Guidelines [1, 2] consider open-chamber instruments only. Guidelines [3] consider both open-chamber and unventillated-chamber instruments. But none of these publications included any AquaFlux™ measurements.

Guidelines [1, 2] discuss (a) person-linked variables, (b) environmental variables and (c) instrumental variables in some depth before presenting their recommendations for best practice. Most of the considerations of sections (a) and (b) are valid for all methods of measurement. The exceptions are section (a) of [2], where the discussion of skin surface temperature includes consideration of how this may affect the measurement probe, and section (b) of both both publications, which include discussions of air circulation and how this may cause fluctuations in open-chamber measurements. Neither of these are concerns for the AquaFlux™. Section (c) and elsewhere is instrument-specific and not applicable to the AquaFlux™.

The table below presents a side-by side comparison of the recommendations of these guidelines with what we recommend for the AquaFlux™.

This question appeared in the title of a poster presentation at the US Symposium of the International Society for Bioengineering and the Skin in Orlando, October 2004 [1]. It concerned in-vitro membrane integrity testing with Franz cells, but the question is equally valid for in-vivo TEWL.

Our answer is that TEWL does indeed give a useful measure of barrier function because a good skin barrier will let through less water than a poor skin barrier.

The difficulty arises because TEWL instruments do not measure TEWL directly. In fact, TEWL instruments are evaporimeters and they measure the flux of water vapour coming from the skin surface. You can interpret this measured water vapour flux as TEWL, if two conditions are satisfied, as follows:

TEWL and nothing but TEWL.

TEWL may not be the only source of water vapour flux. Other possible contributions include perspiration, evaporation of free surface water and leaks in the apparatus. The measured vapour flux can be related to the skin barrier only when these non-TEWL sources have been eliminated.

All must evaporate.

If you’ve satisfied that (1) above is OK, then TEWL will be your only source of water vapour. But remember that TEWL is the migration of condensed water through the epidermis, whereas TEWL instruments measure vapour flux from the surface. All the water arriving at the skin surface must evaporate for the vapour flux to be equal to TEWL. If the humidity in the measurement chamber is too high, then this won’t happen.

The AquaFlux™ is ideal for TEWL measurement, because it maintains a low microclimate humidity at the skin surface, irrespective of ambient conditions. This satisfies condition (2) above better than any other instrument on the market. It also helps with condition (1) by (a) providing the low humidity conditions necessary to get rid of any free surface water quickly, and (b) providing reliable, leak-free means to couple the measurement head to the surface of interest, including Franz cells [2].

References